The innate immune system is an evolutionarily ancient system designed to detect the presence of microbial invaders and activate protective reactions (Beutler, Mol. Immunol. 2004, 40, 845-859). It responds rapidly to compounds that are integral parts of pathogens that are perceived as danger signals by the host. Recognition of these molecular patterns is mediated by sets of highly conserved receptors (van Amersfoort et al., J. Clin. Microbiol. Rev. 2003, 16, 379), whose activation results in acute inflammatory responses. These responses include the production of a diverse set of cytokines and chemokines, directing local attacks against the invading pathogen, and initiation of responses that activate and regulate the adaptive component of the immune system (Dabbagh and Lewis, Curr. Opin. Infect. Dis. 2003, 16, 199-204; Bevan, Nat. Rev. Immunol. 2004, 4, 595-602; Pasare and Medzhitov, Seminars Immunol. 2004, 16, 23-26; Finlay and Hancock, Nat. Rev. Microbiol. 2004, 2, 497-504; Akira et al., Nat. Immunol. 2001, 2, 675-680; Pasare and Medzhitov, Immunity 2004, 21, 733-741).
Evidence is emerging that innate immune responses can be exploited for therapeutic purposes such as the development of adjuvants for vaccines and the treatment of a wide range of diseases including asthma, infections, and cancer. An important concern of such therapies is, however, that over-activation of innate immunity may lead to the clinical symptoms of septic shock (Pittet et al., J. Am. Med. Assoc. 1994, 271, 1598-1601; Rice and Bernard, Annu. Rev. Med. 2005, 56, 225-248). Thus, an important issue for the design of safe immune modulators is a detailed knowledge of structure-activity relationships to harness beneficial effects without causing toxicity.
Lipopolysaccharides (LPS) are structural components of the outer membrane of Gram-negative bacteria and offer great promise for the development of immuno-modulators. LPS consists of a hydrophobic domain known as lipid A, a non-repeating core oligosaccharide and a distal polysaccharide (or O-antigen) (Caroff et al., Microbes Infect. 2002, 4, 915-926; Raetz and Whitfield, Annu. Rev. Biochem. 2002, 71, 635-700). The lipid A moiety of Escherichia coli consists of a hexa-acylated bis-1,4′-phosphorylated glucosamine disaccharide, which has (R)-3-hydroxymyristyl residues at C-2, C-2′, C-3, and C-3′. Both of the primary (3)-hydroxyacyl chains in the distal glucosamine moiety are esterified with lauric and myristic acids, and the primary hydroxyl at the C-6 position is linked to the polysaccharide through a dimeric 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) carbohydrate moiety. It has been proposed that microbial components such as LPS can induce inflammatory responses resulting in tissue damage and alveolar bone loss (Darveau, in Oral Bacterial Ecology The Molecular Basis, ed. Kuramitsu and Ellen, Horizon Scientific Press, Wymond Norfolk, 2000, pp. 169-218).
Recent structural studies have demonstrated that the carbohydrate backbone, degree of phosphorylation, and fatty acid acylation patterns vary considerably among bacterial species (Caroff et al., Microbes Infect. 2002, 4, 915-926; Raetz and Whitfield, Annu. Rev. Biochem. 2002, 71, 635-700; Darveau, Curr. Opin. Microbiol. 1998, 1, 36-42; Erridge et al., Microbes Infect. 2002, 4, 837-851; Alexander and Zahringer, Trends Glycosci. Glycotechnol. 2002, 14, 69-86). Structurally different lipid As may differentially induce proinflammatory responses (Zughaier et al., Infect. Immun. 2005, 73, 2940-2950; Netea et al., Eur. J. Immunol. 2001, 31, 2529-2538; Mathiak et al., Int. J. Mol. Med. 2003, 11, 41-44; van der Ley et al., Infect. Immun. 2001, 69, 5981-5990). For example, in one study, LPS from E. coli O55:B5 induced the production of mediators such as tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 3alpha (MIP-3α) arising from the MyD88-dependent pathway, but caused less production of mediators such as interferon-beta (IFN-β), nitric oxide, and interferon-inducible protein 10 (IP-10) arising from the TRIF-dependent pathway. In contrast, LPS from S. typhimurium invoked strong production of mediators associated with the TRIF-dependent pathway, but caused only minimal production of TNF-α,IL-1↑, MCP-1, and MIP-3α. Heterogeneity in the structure of lipid A within a particular bacterial strain and possible contamination with other inflammatory components of the bacterial cell-wall complicate the use of either LPS or lipid A isolated from bacteria to dissect the molecular mechanisms responsible for the biological responses to specific lipid A molecules. Chemical synthesis of lipid A derivatives has been reported (Erridge et al., Microbes Infect. 2002, 4, 837-851).
Neisseria meningitidis is a Gram-negative bacterium that causes fulminant, rapidly fatal sepsis, and meningitis (Robbins and Freeman, Sci. Am. 1988, 259, 126-133). The morbidity and mortality of meningococcal bacteremia has been linked to a systematic inflammatory response to lipooligosaccharides (LOS) of affected patients (Brandtzaeg et al., J. Infect. Dis. 1989, 159, 195-204; Brandtzaeg et al., J. Infect. Dis. 1992, 166, 650-652; van Deuren et al., Clin. Microbiol. Rev. 2000, 13, 144-166). LOS, a major component of the outer membrane of N. meningitidis, initiates the production of multiple host-derived inflammatory mediators.
Meningococcal LOS lacks the repeating O-antigen of enteric LPS but has a conserved inner core region composed of L-glycero-D-manno-heptosides and a KDO moiety linked to lipid A (Jennings et al., Can. J. Biochem. 1980, 58, 128-136; Gamian et al., J. Biol. Chem. 1992, 267, 922-925). The lipid A of N. meningitidis is hexa-acylated in a symmetrical fashion whereas enteric bacteria have an asymmetrically hexa-acylated lipid A (Darveau, Curr. Opin. Microbiol. 1998, 1, 36-42; Kulshin et al., J. Bacteriol. 1992, 174, 1793-1800; Alexander and Zahringer, Trends Glycosci. Glycotechnol. 2002, 14, 69-86; Erridge et al., Microbes Infect. 2002, 4, 837-851). Also, a number of the fatty acids of N. meningitidis are shorter compared to those of E. coli. At low concentrations meningococcal LOS is a potent inducer of MyD88- and TRIF-dependent cytokines, whereas at the same picomole concentrations E. coli LPS induced comparable levels of TNF-α, IL-1β, and MIP-3α but significantly less IFN-β, nitric oxide, and IP-10 (Zughaier et al., Infect. Immun. 2005, 73, 2940-2950).
Porphyromonas gingivalis is a Gram-negative bacterium implicated in chronic periodontal diseases (Socransky et al., J. Clin. Periociontol., 1998, 25, 134-144). It releases large amounts of outer membrane vesicles containing lipopolysaccharides (LPS), which can penetrate periodontal tissue. Early studies have indicated that P. gingivalis LPS can activate murine macrophages in a TLR2- and TLR4-dependent manner (Darveau et al., Infect. Immun., 2004, 72, 5041-5051). However, it has been suggested that the TLR2 responses maybe due to contaminations with lipoproteins (Ogawa et al., Int. Immunol., 2002, 14, 1325-1332; Ogawa et al., Front. Biosc., 2007, 12, 3795-3812). It has also been found that LPS of P. gingivalis can inhibit IL-6 and IL-1β secretion and ICAM expression induced by enteric LPS by U373 and human peripheral mononuclear cells and human gingival fibroblasts, respectively (Yoshimura et al., Infect. Immun., 2002, 70, 218-225). Another study found that a purified tetra-acylated monophosphoryl lipid A structure can antagonize E-selectin expression in human cells exposed to enteric or P. gingivalis LPS (Reife et al., Cell. Microbiol., 2006, 8, 857-868). It appears that MD2 represents the principle molecular component used by these LPS derivatives for inhibition (Coats et al., J. Immunol., 2005, 175, 4490-4498).
The lipid A moiety of the LPS of P. gingivalis displays considerable heterogeneity and the structures of four compounds have been elucidated, which differ in fatty acid substitution pattern (Ogawa, FEBS Lett., 1993, 332, 197-201; Kumada et al., J. Bacteriol., 1995, 177, 2098-2106). A common structural feature of these derivatives is, however, the presence of unusual branched fatty acids such as R-(3)-hydroxy-13-methyltetradecanic acid and R-(3)-hydroxy-15-methyl hexadecanic acid. The presence of multiple lipid A structures has made it difficult to interpret innate immune responses elicited by P. gingivalis LPS, which in turn has hindered a thorough understanding of the contributions of P. gingivalis LPS to periodontal diseases.
It has long been thought that the inflammatory properties of LPS and LOS reside in the lipid A moiety (Erridge et al., Microbes Infect. 2002, 4, 837-851; Kusumoto, in Molecular Biochemistry and Cellular Biology, Vol. 1, Bacterial Endotoxin Lipopolysaccharides; Chapter 9, Chemical Synthesis of Lipid A, CRC Press, Boca Raton, 1992, pp. 81-105; Kusumoto et al., in Endotoxin in Health and Disease (Ed.: H. Brade), Marcel Dekker, New York, 1999, pp. 243-256; Imoto et al., Tetrahedron Lett. 1985, 26, 1545-1548; Galanos et al., Eur. J. Biochem. 1985, 148, 1-5). Lipid A triggers innate immune responses through Toll-like receptor 4 (TLR4), a member of the TLR family that participates in pathogen recognition. Immediately distal to TLR4 activation are two intracellular cascades that regulate signal transduction processes, gene expression, and production of proinflammatory mediators (Akira et al., Nat. Immunol. 2001, 2, 675-680). One of these cascades requires a specific intracellular adaptor protein called MyD88, while the other cascade utilizes the TRIF adaptor protein. The MyD88-dependent pathway leads to up-regulation of cytokines and chemokines such as TNF-α, IL-1β, IL-6, and MCP-1, whereas the TRIF-dependent pathway leads to the production of IFN-β, which in turn activates the STAT-1 pathway resulting in the production of mediators such as IP-10 and nitric oxide (Karaghiosoff et al., Nat. Immunol. 2003, 4, 471-477).
However, recent studies have shown that lipid A expressed by meningococci with defects in KDO biosynthesis or transfer has significantly reduced bioactivities compared to KDO2 containing meningococcal LOS (Zughaier et al., Infect. Immun. 2004, 72, 371-380). Removal of the KDO moieties of wild type LOS by mild acetic acid treatment also attenuated cellular responses. Interestingly, dendritic cells stimulated with KDO2-lipid A from meningococci but not lipid A alone stimulated naïve allogeneic CD4+ cells to secrete enhanced levels of IFN-γ relative to T-cells primed with immature dendritic cells (Zughaier et al., Vaccine 2006, 24, 1291-1297).
Several other studies have suggested that the KDO moiety of LPS or LOS contributes to inflammatory responses. For example, it has been found that salmonella lipid A is inactive whereas the parent LPS is a potent activator of NF-κB in a TLR4-dependent manner in a human monocytic cell line (Muroi and Tanamoto, Infect. Immun. 2002, 70, 6043-6047). In addition, a synthetic enteric lipid A containing a di-KDO moiety was a more potent elicitor of TNF-α and IL-6 compared to its parent lipid A (Yoshizaki et al., Angew. Chem. Int. Ed. 2001, 40, 1475-1480; Yoshizaki et al., Angew. Chem. 2001, 113, 1523-1528). Furthermore, LPS from a nitrogen-fixing symbiont, Rhizobium sin-1 is able to significantly inhibit the E. coli LPS-dependent synthesis of TNF-α by human monocytic cells (Demchenko et al., J. Am. Chem. Soc. 2003, 125, 6103-6112; Santhanam et al., Chem.-Eur. J. 2004, 10, 4798-4807; Lee et al., Chembiochem 2006, 7, 140-148; Zhang et al., Bioorg. Med. Chem. 2007, 15, 4800-4812; Vasan et al., Org. Biomol. Chem. 2007, 5, 2087-2097). A comparison of the biological responses of synthetic and isolated lipid A derivatives and R. sin-1 LPS indicated that the KDO moieties are important for optimal antagonistic properties. Thus, it is probable that the cell surface receptors that recognize LPS bind to the lipid A as well as to the KDO moiety of LPS.
Several studies have reported compounds that can antagonize cytokine production induced by enteric LPS (Rossignol et al., in Endotoxin in Health and Disease, eds. Brade et al., Marcel Dekker, Inc., New York, 1999, vol. 1, pp. 699-717). Most efforts have been directed towards the synthesis of analogs of lipid A of Rhodobacter sphaeroides (Christ et al., J. Am. Chem. Soc., 1994, 116, 3637-3638; Christ et al., Science, 1995, 268, 80-83) and derivatives of lipid X (Golenbock et al., J. Biol. Chem., 1991, 266, 19490-19498; Lam et al., Infect. Immun., 1991, 59, 2351-2358; Kawata et al., in Novel Therapeutic Strategies in the Treatment of Sepsis, ed. Morrison and Ryan, Marcel Dekker, New York, 1995, pp. 171-186; Peri et al., Angew. Chem. Int. Ed., 2007, 46, 3308-3312). Analogs of the lipid A moiety of Helicobacter pylori have also been shown to inhibit IL-6 secretion by human whole blood cells (Fujimoto et al., Tetrahedron Lett., 2007, 48, 6577-6581). Recently, it was reported that synthetic lipid As derived from Rhizobium sin-1 can prevent the induction of TNF-α by E. coli LPS in human monocytic cells (Demchenko et al., J. Am. Chem. Soc., 2003, 125, 6103-6112; Santhanam et al., Chem.-Eur. J., 2004, 10, 4798-4807; Lee et al., Chembiochem, 2006, 7, 140-148; Zhang et al., Bioorg. Med. Chem., 2007, 15, 4800-4812; Vasan et al., Org. Biomol. Chem., 2007, 5, 2087-2097).
Although studies with LOS isolated from meningococci have indicated that it possesses unique immunological properties, heterogeneity in the structure of lipid A and possible contaminations with other inflammatory components of the bacterial cell wall have made it difficult to confirm these results (Zughaier et al., Infect. Immun. 2005, 73, 2940-2950; Zughaier et al., Infect. Immun. 2004, 72, 371-380; Zughaier et al., Vaccine 2006, 24, 1291-1297). Furthermore, the acylation patterns, as well as fatty acid length, differ between meningococcal and E. coli lipid A. Hence, it has been impossible to establish which structural feature is responsible for the unique inflammatory properties.